Past investigation has demonstrated that a significant fraction of puruvate kinase (Type L) present in hepatocytes from fed and fasted rats is not subject to hormonal regulation by phosphorylation/dephosphorylation mechanisms. We have now developed cell culture techniques which indicate that the modifications leading to loss in regulation are the consequence of post-translational modification of the enzyme. When pyruvate kinase is newly synthesized in fresh hepatocytes, it appears as one predominant isoelectric form which is phosphorylated in response to glucagon. As hepatocytes are maintained in culture for up to two days, multiple isoelectric forms of the enzyme which can be separated in 8 M urea are generated. These modified isoelectric forms do not appear to be regulated by phosphorylation and the native enzyme loses the ability to be regulated by glucagon. Our studies also indicate that generation of non-phosphorylatable forms of the enzyme is modulated by hormonal (adrenal glucocorticoids) and nutritional factors in a mechanism which remains to be elucidated. The nature of the posttranslational regulation of pyruvate kinase also has not yet been established, although proteolysis is suspected to be responsible for generation of the multiple isoelectric forms of the enzyme observed in 8 M urea. The objectives of the proposed investigation are to further characterize the regulation of pyruvate kinase by nutrients, glucocorticoids and other hormones. Five specific aims are outlined: 1. Determine the chemical basis for the modifications. 2. Characterize the influence of glucagon, insulin, and nutritional factors on generation of multiple isoelctric forms of the enzyme in cultured hepatocytes. 3. Quantify the amount of various isoelectric forms present in liver from intact rats. 4. Characterize the punitive proteases involved in this regulation. 5. Complete present studies on the kinetic and physical properties of purified liver pyruvate kinase. The completion of these studies should significantly contribute to our understanding of the role of pyruvate kinase in the regulation of hepatic carbohydrate metabolism since it is believed to be an important target enzyme of several hormonal and nutritional effects on the liver.